The majority of analytical cytology is conducted using highly sophisticated instruments such as fluorescence microscopes and flow cytometers to examine, count, or otherwise analyze cells stained with fluorescent dyes. Without fluorescent calibration standards, it is not possible to make quantitative determinations about the cells with respect to their fluorescence intensities.
With fluorescence microscopes, it would be useful to have fluorescent calibrated particles which have the same size and fluorescent spectral properties as the stained cells. Such particles could be used to optimize the alignment of the microscope. If such particles had calibrated numbers of fluorescent molecules on them, they could be used as an internal quantitative standard, i.e., mixed with the cells on a slide to compare the brightnesses of cells and particles. Quantitative estimates of the fluorescence intensity of the cells could be made by eye, or accurate measurements could be made with photocells attached to the fluorescent microscope.
Flow cytometry is the process of analyzing and sorting cells in a flowing stream. This is accomplished by intersecting the stream with an incident light, usually a laser, and detecting the resulting scattered light and fluorescence of the individual cells as a function of the particular physical characteristics or attached fluorescent dye, respectively. In addition, electronic volume sensing has also been incorporated in some flow cytometry instruments. With this array of detectors, sub-populations of cells can be analyzed and sorted in arbitrary terms by just detecting their qualitative differences. However, without size and fluorescent standards, no quantitative information on the individual cells can be gained other than the number of them counted and their proportion relative to the rest of the sample.
To determine quantitative differences between subpopulations of cells, and moreover, to give individual populations a quantitative relevance, standards are necessary with known amounts of fluorescence to which these cell samples can be compared. In FIG. 1, a microbead containing a fluorescent dye, fluorescein isothiocynate (FITC), is shown along with a cell labeled with the same dye. If a series of such microbeads containing varying amounts of the fluorescent dye is run on a flow cytometer, the resulting distributions will be obtained, as shown in FIG. 2, indicated by "Bead 1, Bead 2, and Bead 3". Now if a cell population stained with the same dye is also run on the flow cytometer under the same conditions, then the fluorescence intensity of the cells can be quantitatively compared to those of the calibrated microbeads.
Various fluorescent particles have been used in conjunction with flow cytometry including fixed cells, pollen, fluorescent microbeads, and stained nuclei. However, their use has been limited for the most part to instrument alignment and size calibration. Quantitation of fluorescence intensity of cell samples has been hampered by not having a highly uniform, stable particle which has the same excitation and emission spectra as the cells being measured. Those particles which contain the proper dyes, e.g., the fluorescein stained nuclei (marketed as Fluorotrol-GF by Ortho Diagnostic Systems, Inc.) are not stable over long periods of time and those which are considered stable, e.g., the microbeads, have not contained the same dyes which stain the cells. Highly uniform fluorescent microbeads have been available from various sources for a number of years. However, none of these beads have been suitable as quantitative standards for flow cytometry instruments because (1) many of these fluorescent microbeads are smaller than the cells to be analyzed and (2) the fluorescent dyes which have been incorporated into the small microbeads are different from those attached to the cells and (3) beads which are the preferable size (about 4-10 microns in diameter) have been expensive to produce and difficult to obtain because the techniques used in their production give particles of wide variability in size, shape, and other physical properties.
Attempts have been made to cross-calibrate microbeads containing one dye against solutions or cells containing a different dye, e.g., cumerin containing microbeads against fluorescein solutions. However, such a calibration is only good for the one excitation and emission filter system. Use of slightly different filter systems, which may occur with instruments from different manufacturers, can significantly alter the quantitative results. As recognized in parent application Ser. No. 685,464, the key to having a useful fluorescent standard which can be used on any instrument or filter system is for the microbead to have the same excitation and emission spectra as the sample. A more subtle point recognized by the invention is that the environment of the dye molecules can have a large effect on the fluorescence spectra. This is demonstrated in FIG. 3, where the emission intensity of a cell labeled with fluorescein is compared to that of microbeads with fluorescein on the surface and fluorescein within the body of the hydrophobic microbead. The surface fluorescenated microbead has the fluorescein in contact with the aqueous medium, and has the same emission properties as a function of wavelength as does the fluorescein labeled cell suspended in an aqueous medium, whereas, the microbead with the fluorescein within the hydrophobic bead body has a very different response because both the excitation and emission spectra have shifted and broadened as a function of the dye being in a hydrophobic medium and not in contact with water. The related spectra as recognized by the invention are shown in FIG. 4.
Recently, larger microbeads suitable for size calibration of biological cells have been synthesized in outer space (see NASA TM 78132 "Large-size Monodisperse Latexes as a Commercial Space Product", and U.S. Pat. No. 4,247,434). In addition, U.S. Pat. No. 4,336,173 to Ugelstad discloses a method for preparing uniform microbeads of a size suitable for flow cytometry calibration in a conventional, earth-bound laboratory. However, none of these large microbeads were reported to contain a fluorescent dye. Moreover, synthesis as described in U.S. Pat. No. 4,336,173 was found to be hampered by agglomeration and high doublet formation. Even with the suggested polymeric stabilizers, the yields of mono-dispersed microbeads were lower than acceptable. The present invention has for its object, among others, the accomplishment of this task.
In the Ugelstad method, a dispersion of small, uniform, seed particles produced by conventional means is contacted with a first swelling agent which is absorbed into the particles to cause some swelling of the particles. Subsequently, a second swelling agent, usually a polymerizable monomer, is likewise absorbed into the particles. Upon internal polymerization of this monomer, the particle undergoes additional swelling. The desired particle size is reached by absorbing pre-calculated amounts of the first and second substances into the microparticle.
Ugelstad teaches that, when using an oil soluble polymerization initiator which is somewhat soluble in water, the initiator may be added after the monomer has diffused into the particles or it may be dissolved in the monomer before the latter diffuses into the particles (see Ugelstad; at column 6, line 65 to column 7, line 2). Ugelstad further teaches that, when using oil soluble initiators which are less water soluble, it will be necessary to add the initiator together with the first swelling agent (see column 7, lines 2-5). When manufacturing beads, however, adding the initiator with the first swelling agent is disadvantageous because the initiator is unstable and swelled beads containing it must be handled carefully and used quickly. By not including the initiator in the first swelling agent the manufacturer can make a large stable batch of swelled seed beads which can be stored for years, giving the manufacturer the ability to choose the initiator and second swelling agent flexibly while using the same swelled seeds, and enabling the manufacturer to get more reproducible batches of finished polymerized beads by allowing him to draw from a uniform stock solution of swelled beads. At the same time, however, it is desirable to use less water soluble initiators while manufacturing beads, as such initiators are less likely to cause agglutination among the beads and decrease the yield of monodisperse beads. In other words, the prior art teaches that, in order to get good yields of monodisperse beads, an initiator having a very low solubility in water should be used. However, the prior art further teaches that, when such an initiator is used, it should be added with the first swelling agent, and this requirement deprives the manufacturer of the advantages set forth above.
It is therefore an object of this invention to provide a method of manufacturing relatively large polymeric microbeads so that there is a very high yield of monodisperse beads without requiring the manufacturer to add the initiator to the seed beads at the same time the first swelling agent is added. Other objects will appear as the description proceeds.